Cell Differentiation, 4 (1976) 415--427 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

415

R N A S Y N T H E S I S IN T H E DROSOPHILA M E L A N O G A S T E R P U F F S

E.S. BELYAEVA and I.F. ZHIMULEV

Institute of Cytology and Genetics, Novosibirsk, 630090, U.S.S.R. Accepted 27 October 1975

Autoradiographical analysis of [3H]-uridine incorporation into three large puffs of Drosophila melanogaster has revealed that after 1 rain pulse with isotope the label is observed above the whole puff area. When puffs were induced by high temperature, RNA synthesis was sharply increased (or started) in many sites of the developing puff. No significant delay in the transport of newly-synthesized RNA in large puffs, in comparison with small ones, has been found. In regions without temperature-induced puffs RNA synthesis and its transport are apparently delayed under influence of heat shock.

Processes of p r o m i n e n t p u f f f o r m a t i o n have been studied intensively at a m o r p h o l o g i c a l level (Melchelke, 1 9 6 1 ; B e e r m a n n , 1 9 6 2 ; Panitz, 1 9 6 4 , 1 9 6 5 ; Berendes, 1 9 6 5 ; K i k n a d z e , 1 9 7 1 ; A s h b u r n e r , 1 9 7 2 ; Pelling, 1972; Ber e n d e s et al., 1 9 7 4 ; Belyaeva et al., 1 9 7 4 ; Zhimulev, 1974). H o w e v e r , the regularities o f R N A a c c u m u l a t i o n and d i s t r i b u t i o n o f labelled R N A precursors in p u f f s are far f r o m being clear; i n d e e d the available results are contrad i c t o r y (Berendes, 1 9 6 8 ; B e r e n d e s et al., 1 9 7 4 ; Ellgaard et al., 1971). This p a p e r will deal with d i s t r i b u t i o n o f label a f t e r i n c o r p o r a t i o n o f [3H]-uridine into some large c o m p l e x puffs in D. melanogaster c h r o m o somes. T h e i n t e n s i t y of a c c u m u l a t i o n and r e d i s t r i b u t i o n o f n e w l y synthesized R N A in such puffs will also be discussed. MATERIALS AND METHODS P r e p u p a e (99) o f a wild l a b o r a t o r y stock, B a t u m i - L , and third instar larvae (9?) h e t e r o z y g o u s f o r t w o m u t a n t alleles at the giant (gt: 1--0.9) locus (gtE~/gt 13z) have been used. T h e c h r o m o s o m e s o f the mutant giant are 2--4 times t h i c k e r t h a n c h r o m o s o m e s o f wild t y p e stocks (Bridges, 1 9 3 5 ) . T h e alleles of gt used were o b t a i n e d f r o m and d e s c r i b e d b y K a u f m a n (1972). Larvae were m a i n t a i n e d o n s t a n d a r d m e d i u m - - g t at 20°C, B a t u m i - L at 25 ° C. Puffs were i n d u c e d b y transferring isolated salivary glands into Ephrussi--Beadle saline at 37°C. P r e p u p a e were i n j e c t e d w i t h 0.1 pCi o f [3H]-uridine (49 Ci/mM, Amersham) per animal. In o t h e r cases isolated glands were i n c u b a t e d in vitro for

416 various times in medium containing [3HI-uridine (100 pCi/ml or 1000 pCi/ ml). Procedures for chromosome staining and the preparation of slides have been described previously (Zhimulev et al., 1975). The time of exposure of the emulsion was chosen so that the number of silver grains above the puffs to be studied was similar in comparable experiments (about 70--90 grains above large puffs in experiments quantitatively analyzed). 70--100 nuclei from 6--12 animals have been analyzed in each experimental run. Chromosome regions were identified from the revised maps of C.B. and P.N Bridges (in: Lindsley et al., 1968). RESULTS

Puffs having maximal size in O-hr prepupa Relative radioactivity of large and small puffs after various intervals o f [3H]-uridine administration To establish whether accumulation of the newly synthesized R N A differs between large and small puffs, the number of silver grains above large puffs (71CE, 85F, 88D) was related to that above small puffs (6lAB, 72AB, 86EF) and these ratios were compared after brief (5 min) and long (30 min) periods of [3H]-uridine incorporation (Table I). The morphology and size of these puffs in the Batumi-L stock, were described earlier (Zhimulev, 1974). When making such a comparison, it is necessary to be sure that the activity of these regions is not changing for 30 min. The relative intensity of label incorporation into the puffs, after a 5 min pulse appears to be the same in both 0-hr prepupa and in 25-min prepupa (Table I). Thus, the activity of these regions is constant during this interval. Our data are confirmed by Ashburner's morphological findings (Ashburner, 1972). If newly synthesized R N A accumulates in large puffs (as suggested by Ellgaard et al., 1971) then the relative radioactivity of large puffs should increase with longer times of [ 3H]-uridine incorporation. However, the relative labelling of the large puffs after 30 min [3H]-uridine administration is, at maximum, 50% higher than that after 5 min. The results differ significantly only in one experimental run (Table I). The data are similar both in vivo and in vitro, indicating that the synthetic activity of the regions examined does not change in isolated glands for 30 min. Distribution of silver grains above large puffs Three large puffs, having maximal size in 0-hr prepupae (63DE-5, 71CE, 82EF) were chosen for analysis. These puffs appear to be formed from many bands and are complex (Fig. 1). After incubation of glands with [3H]-uridine for 1 min the label is seen over the whole area of the puffs (Fig 2). The pattern of label location does not change if the incubation time is increased. 'Strips' of silver grains, like those observed by Berendes in puff 2-48C (Berendes, 1968), were only found above puffs which labelled very weakly

0 0 25

0 0 0 25

Injection in vivo, 0.1 pCi/larva

Incubation of glands in vitro, 1000 pCi/ml

* Standard error. ** Results differ significantly.

Prepupae ages at moment [3H]uridine administration (min)

Conditions of [SH]-uridine incorporation

1 5 30 5

5 30 5

Exposure to [3H]uridine (min) 71CE/ 6lAB

7.2±0.4 9.6±0.9 6.0±0.4 5.9±0.3 6.2±0.3 7.7±0.6 7.0±0.3

7 1 CE/ 7 2 A F

6.3**±0.2* 8.8**±0.3 5.9**±0.3 6.3±0.3 6.4±0.5 8.0±0.4 7.0±0.4

1.0±0.1 1.2±0.1 1.1±0.1 1.1±0.1

1.0±0.1 1.2±0.1 1.1±0.1

72AF/ 6lAB

1.1 1.4 1.1 1.1

1.3 1.4 1.3

85F/ 86EF

Adjusted ratios of grain number over regions:

0.7 0.8 0.7 0.7

0.7 0.8 0.9

85F/ 88D

0.6 0.6 0.6 0.6

0.6 0.5 0.6

86EF/ 88D

Intensity of [3H]-uridine incorporation into chromosome regions with large puffs in relation to chromosome segments with small puffs.

TABLE I

b.a

418

e

;

6} C

i E

'I]

71 B C

Fig. 1. Relative intensity of [3H]-uridine incorporation into different areas of puffs in Batumi-L chromosomes, a) Chromosome regions according to Bridges. b) Puff morphology in chromosomes of 0-hr prepupae, c ~ ) Distribution of silver grains over puff areas (in % of total grain number over puff): c) glands of 0-hr prepupae incubated with [3H]uridine for 5 rain; d) glands of 0-hr prepupae incubated with [SH]-uridine for 30 min; e) glands of 25 min prepupae incubated with [3H]-uridine for 5 rain.

and such label p a t t e r n s were i n d e p e n d e n t o f the time o f e x p o s u r e to [3H]uridine a n d were e x t r e m e l y rare. T o a n a l y z e f u r t h e r the silver grain d i s t r i b u t i o n above large p u f f s each p u f f was divided into three areas. T h e p a t t e r n o f relative r a d i o a c t i v i t y in these areas was similar after 5 and 30 min [3H] -uridine i n c o r p o r a t i o n and did n o t d e p e n d on w h e t h e r a p u f f h a d c o n s t a n t size during this p e r i o d ( 7 1 C E ) o r was halved in size ( 6 3 D E - 5 and 8 2 E F ) . It is o f interest t h a t , w h e n [3H]-uridine is i n c o r p o r a t e d into c h r o m o s o m e s o f 25-min p r e p u p a e , i.e. at the m o m e n t o f

419

a

b

Fig. 2. Labelling pattern of puffs 63DE--5 (a), 71CE ~b), 82EF (c) in chromosomes of 0-hr Batumi-L prepupae after incubation of glands with [ H]-uridine for 1 rain.

regression of puffs 63DE-5 and 8 2 E F (Ashburner, 1972), then the relative intensity of labelling is similar to that in maximal-sized puffs (Fig. 1). Thus, we have found neither any considerable R N A accumulation into large puffs as compared to small ones, nor visible redistribution of label among various sites within complex puffs.

Puffs induced by temperature 37°C Location o f R N A synthesis in arising p u f f Puff 63B gtE6/gt 132 larvae already show decondensation of some bands in the 63B region during the first minute of exposure to 37°C. A remarkable puff has formed within 2 min. No other bands become involved in this puff, it only increases in size, reaching a maximum after 5 min temperature treatment (Fig. 3). When salivary glands were placed into Ephrussi--Beadle saline containing [3H]-uridine at 37 ° C for one minute the label was located above the area of all bands involved in puffing in the 63B region (Fig. 4). This is especially well seen if the chromosome is slightly stretched (Fig. 4d). The location of label does not depend on the time of incubation with [3H]uridine at 37°C (1 min or 30 min). Puffs 87A and 87B These puffs are formed slightly slower than 63B. 87A reaches its maximal size within 10 min, and 87B within 15--20 min after the beginning of heat shock (Fig. 5). Similar data have been obtained by Ellgaard and Clever (1971). Nevertheless, even by the end of the first minute of treatment the areas of intensive transcription are clearly revealed in both puffs and their location is unchanged with longer temperature shock and incorporation (Fig. 6).

420 a

-63£--

B

0

Fig. 3. Heat-shock induction of puff 63B in the chromosomes of gtF-~6/gt 13z larvae; glands were heat shocked: b) 1 min, 37°C; c) 1.5 min, 37°C; d) 2 min, 37°C;e) 10 min, 37°C; a) regions 63B according to Bridges.

Fig. 4. [3H]-uridine incorporation into region 63B in chromosomes of gtF~6/gt 13z larvae. • • ~ a) Control, no heat-shock; b) 37 0 C + [ 3 H ] - u r l- d m e , 1 mm; weak ~ labelhng; c) 37 0 C + [JH]-uridine, 1 min; 'strong' labelling; d) 37°C + [~H]-uridine, 1 rain; 'strong' labelling of a slightly stretched c h r o m o s o m e ; e ) 37°C + [3H ]-uridine, 30 rain.

421

Fig. 5. Heat-shock induction of puffs 87A and 87B in the chromosomes of gtE~/gt13z larvae, a) Region 87AB according to Bridges; b) Region 87AB in control, no heat treatment;c--f) Exposure of glands to heat (37 °C): c) 1 min;d) 1.5 min;e) 5 rain; f) 15 min.

a

•

~

b

C

Fig. 6. [3H]-uridine incorporation into puffs 87A and 87B. a) 37°C + [SH]-uridine, 1 min; b) 37°C + [3H]-uridine, 3 min; c) 37°C + [3H]-uridine, 30 min; d) 37°C, 25 min; then 37°C + [3H]-uridine, 3 min. areas o f intensive t r a n s c r i p t i o n are clearly revealed in b o t h p u f f s and their l o c a t i o n is u n c h a n g e d w i t h l o n g e r t e m p e r a t u r e s h o c k and i n c o r p o r a t i o n (Fig.

6). Relative intensity of [SH]-uridine incorporation into regions with temperature-induced puffs and into regions without such puffs T h e relative r a d i o a c t i v i t y o f the p u f f s 63B, 8 7 A , 87B increases as m u c h as 4--6 times d u r i n g t h e first 5 m i n of i n d u c t i o n (Table II). A f t e r 30 m i n in-

3.38+0.25

4.74+0.27

4.76+0.55

1 2 . 5 1 + 1.72

5 min after transfer of glands t o 37°C a n d [3 H ]-uridine 1000/~Ci/ml

30 m i n a f t e r t r a n s f e r o f glands t o 37°C a n d [3 H ] - u r i d i n e 100 p C i / m l

30 m i n a f t e r t r a n s f e r o f glands to 37°C a n d [3 H ] - u r i d i n e 1000 p C i / m l

30 m i n a f t e r t r a n s f e r o f glands t o 37°C a n d 5 min after transfer to 37°C a n d [3H]uridine, 1000 pCi/ml

* S t a n d a r d error.

0.53-+0.05"

63B/61AB

24.84-+3.48

10.26-+0.85

9.65+0.56

9.29-+0.75

1.83-+0.15

63B/ 62F + 63A

2.41+0.29

2.66-+0.23

2.41+0.17

3.66+0.34

3.91-+0.30

6lAB/ 62F + 63A

A d j u s t e d ratios o f grain n u m b e r over regions:

Control: glandincubat i o n w i t h [ 3 H ]-uridine, 1000 pCi/ml, 5 m i n

E x p e r i m e n t a l runs

15.76 + 1.69

3.31.+-0.29

3.15+0.32

2.35-+0.18

0.56 + 0 . 0 6

87A/86AD

4.24+0.33

1.22-+0.09

--

0.84-+0.05

0.12 + 0 . 0 1

87A/ 87D--88C

30.37-+3.46

7.28 + 0 . 5 3

8.61 + 1.10

2.53 + 0 . 1 8

0.75+0.06

87B/86AD

8.03-+0.61

2.54 -+0.19

--

0.84-+0.06

0.18-+0.02

87B/ 87D--88C

0.36A0.04

0.40 + 0 . 0 4

--

0.33-+0.02

0.27 + 0 . 0 2

86AD/ 87D--88C

I n t e n s i t y of [ 3 H ] - u r i d i n e i n c o r p o r a t i o n into c h r o m o s o m e regions o f gtF'6/gt 13z larvae w i t h t e m p e r a t u r e - i n d u c e d p u f f s in r e l a t i o n to c h r o m o s o m e s e g m e n t s w i t h o u t such puffs,

T A B L E II

423 cubation with [3H]-uridine at 37°C the relative intensity of labelling of the puffs 63B and 87A appears to be the same while it has increased in 87B (Table II). This difference seems to be due to the slower formation of 87B compared to 87A and 63B. On the other hand in all three puffs the intensity of [3H] -uridine incorporation has increased by the end of 30 min incubation as compared to regions w i t h o u t temperature-induced puffs: in glands exposed to [3H] -uridine for 5 min after 25 min of heat pretreatment labelling was 3--4 times higher than after 5 min of incorporation of [3H]-uridine at 37 ° C w i t h o u t pretreatment. Thus, the relative a m o u n t of label accumulated in puffs during 5 and 30 min is similar, though the relative intensity of [3H]-uridine incorporation differs greatly in them at the very beginning and at the very end of the heat stock. These findings are in contrast to those obtained for normal puffs of 0-hr prepupa, therefore a special experiment has been carried out on the temperature-stimulated p u f f 63B. The results are given in Fig. 7, and confirm the great increase in relative intensity of label incorporation into this puff during heat treatment; the relative a m o u n t of label incorporated for 3 and 30 min being constant. Estimation of silver grain number over regions (in 200 nuclei from 10 animals) not containing any heat-shock induced puffs have shown that the intensity of label incorporation into such regions decreases during the heat treatment. Thus, mean grain numbers over l A B and 6 l A B , when [3H]-uridine was incorporated during the first 3 min of heat were 10.0 + 0.5 and 20.5 + 1.1 respectively whereas after 25 min of pre-

i

A

10I|

4

12 20

1 0 ~

2

4

30F ~--~

28

6

8

E 10 F

B

lO~, X ! 4 12

30I _~_2

4

2

4

30t L 10

D I

12

G

C 30

4

~

10

\

2 Fig. 7. Relative intensity of [SH]-uridine incorporation into puff 63B. Abscissa: Ratios of silver grain number over regions 63B/61AB (a--d) and 63B/66F (e--h). Ordinate: chromosomes number, a,e) 37°C, 25 rain; then 37°C + [3H]-uridine, 3 min; b,f) 37°C, 12 min; then 37°C + [SH]-uridine, 3 min; c,g) 37°C + [SH]-uridine, 3 min;d,h) 37°C + [SH]-uridine, 30 min.

424

treatment plus 3 min [3H]-uridine at 37°C the respective numbers were 2.9 + 0.2 and 4.8 + 0.3. These results are shown in Fig. 8, where it is clearly seen that in the case of 25 min of heat pretreatment the radioactivity of induced puffs is much higher compared to other loci, including the nucleoli. In this experiment all the regions having a very high intensity of [3H] -uridine incorporation after heat shock are distinctly revealed. There are as follows: 2E1--2, 5C3--16, 10B3--16, 10EF, 18D3--13, on the X-chromosome; 22E1--2, 26A3--9, 24D3--11, 37EF, 38F1--6 on 2L; 43E3--18, 44DE, 45E1--2, 47B1--8, 47D1--4, 48E3--12, 49E1--15, 50C3--7, 50D1--7, 51D3--12, 56E1--6 on 2R; 61C1--4, 63B, 64E3--13, 67AB, 70B1--7, 73D1--7, 78E1--2 on 3L; 84E1--6, 85B6-9, 87A7--10, 87B10--15, 88E3-5, 93D1--10, 95D1--11 on 3R. Many of these puffs are induced by heat a,

....

Fig. 8. Labelling pattern of chromosomes and nucleoli after heat shock, a) 37°C + [SH]uridine, 30 rain; b, d) 37°C + [SH]-uridine, 3 min; c, e) 37°C, 25 r a i n ; t h e n 37°C + [SH]uridine, 3 min.

425

shock, anaerobiosis, saline (Ashburner, 1970) and transplantation of salivary glands into imaginal a b d o m e n a (Staub, 1969). DISCUSSION

Area o f transcription in complex puffs Our data on uridine incorporation into complex puffs at the time of their maximal size stated above seem to show 'polylocality' of labelling: after exposure to uridine for 1 min silver grains are distributed over whole puffs. This may result from the spread of the DNP of the puff band as it uncoils, or from real 'polylocality' of R N A synthesis in the complex puff. The pattern of [3H]-uridine incorporation into puffs at the first minute of their induction is, from our point of view, the main argument favoring the suggestion that transcription begins simultaneously in all bands forming puff. Regions adjacent to puffs remain 'weakly' labelled even when the puffs are very intensively labelled. This proves that the limits of the area of 'strong' incorporation are n o t distorted by scattering of H-particles. This is also supported by lengthening of the transcription area of a puff in slightly stretched chromosomes: it means the label belongs to all of bands constituting a puff. Thus, R N A synthesis appears to begin or greatly increase during one minute at several sites of a complex puff. Experiments with high resolution autoradiography are needed for a further analysis of this phenomenon. At present, we can suppose that complex puffs, at least in the cases mentioned above, presents a system of transcription units having a c o m m o n reaction to an inductor. This opinion is in agreement with the suggestion that puffs can produce transcripts different 'with respect to their information content' (Mechelke, 1961; Berendes et al., 1974). It is unclear, however, which part of the R N A transcribed from complex puffs is transferred to a cytoplasm and whether genes located within one puff are functionally related to each other.

On accumulation o f newly-synthesized R N A in puffs Our findings are in contrast with the suggestion of Ellgaard and Clever (1971) that the rate of R N A synthesis in large puffs is not higher than in small puffs b u t that its processing is delayed resulting in accumulation of R N A in these regions. Firstly, the rate Of R N A synthesis in temperature-induced puffs is obviously increased by as many as 3--4 times within the first minutes of heat induction. Similar results are given in Berendes' paper (1968). Should the increase of the rate of R N A synthesis be accompanied b y delayed transport the puffs would go on increasing in size for a long time. However, such a p u f f as 63B attains its maximal diameter within 5 min of heat shock. Data on rela-

426 tive intensity of puff labelling in 0-hr prepupa support the idea that the accumulation of newly-synthesized R N A is similar in large and small puffs. No visible redistribution of this RNA between particular puff areas has been found. The latter results are not similar to those of Berendes (1968), though this m a y result from the different experimental conditions used. Interpretation of the labelling pattern resulting from heat shock is more difficult. In two independent experiments we found that pretreatment at 37°C followed by pulse of [3H]-uridine increased the intensity of label incorporation into temperature-induced puffs compared with that in regions without such puffs. However, the amount of label incorporated throughout the period of treatment (30 min) appeared to be the same as after 3 min of induction. The most reasonable interpretation of these data, from our standpoint, is as follows. The intensity of RNA synthesis sharply increases in sites of temperatureinduced puffs, attaining its maximum within the first 5--10 min of induction, after which RNA synthesis and transport is balanced. In the other chromosome regions intensity of synthesis progressively declines, resulting in an increase of labelling of temperature-induced puffs at extended temperature treatment. Along with this the transport of newly synthesized RNA is delayed in chromosome regions possessing no temperature-induced puffs, therefore if [3H]-uridine is incorporated throughout 30 min after onset of heating, radioactivity of these regions proves to be high. If so, it is possible to believe that in all chromosome regions without temperature-induced puffs the rate of RNA synthesis is greatly decreased following temperature rise, at least in vitro. There seem to be two kinds of loci in D. melanogaster chromosomes which react quite differently to heat-shock. This opinion is contrary to that in Ellgaard and Clever's paper (1971), but nevertheless it is also in a good agreement with their data showing that the total a m o u n t of R N A synthesized in the nucleus does not increase after temperature induction of puffs and that the synthesis of pre-r-RNA and processing of Hn-RNA are delayed by the heat shock. Our suggestion is also supported by Ashburner's finding (1970) that many Drosophila puffs regress under high temperature action. Disturbance of normal chromosome functioning by temperature shock is witnessed by the fact that even 4 hr after its end the induction of puffs with ecdysone is abnormal. Moreover heat treatment of larvae gives rise to a significant disturbance of development, which is visualized in the numerous and different morphoses of the imagos (Ashburner, 1970). ACKNOWLEDGEMENTS We are grateful to Prof. Dr. L.I. K o r o c h k i n and Dr. I.S. G u b e n k o for reading the manuscript and for making helpful suggestions. T h a n k s are due to Dr. T.C. K a u f m a n for providing the giant stocks.

427 REFERENCES Ashburner, M. : Chromosoma 3 1 , 3 5 6 - - 3 7 6 (1970). Ashburner, M.: In: Results and Problems in Cell Differentiation, Vol. 4; ed. W. Beermann (Springer-Verlag, Berlin--Heidelberg--New York) (1972). Beermann, W. : Protoplasmatologia, VI-D (Springer-Verlag, Vienna) ( 1962). Belyaeva, E.S., L.S. Korochkina, I.F. Zhimulev and N.K. Nazarova: Tsitologia (U.S.S.R.) 1 6 , 4 4 0 - - 4 4 6 (1974). Berendes, H.D. : Chromosoma 17, 35--71 (1965). Berendes, H.D.: Chromosoma 24,418--437 (1968). Berendes, H.D., C. Alonso, P.J. Helmsing, H.J. Leenders, J. Derksen: Cold Spring Harbor Symp. Quant. Biol. 3 8 , 6 4 5 - - 6 5 4 (1974). Bridges, C.B.: J. Heredity 26, 6064 (1935). Ellgaard, E.G. and U. Clever: Chromosoma 36, 60--78 (1971). Kaufman, T.C. : Genetics 71, 28--29 (1972). Kiknadze, I.I.: In: Advances in Current Genetics, 3, ed. N.P. Dubinin (Nauka, Moskow) (1971). Lindsley, D.L. and E.H. Grell: In: Genetic Variation of Drosophila melanogaster. Carnegie Inst. Wash. Publ., N 627 (1968). Mechelke, F.: Naturwissenschaften 48, 29 (1961). Panitz, R. : Biol. Zentralblatt 8 3 , 1 9 7 - - 2 3 0 (1964). Panitz, R. : Chromosoma 1 7 , 1 9 9 - - 2 1 8 (1965). Pelling, C.: In: Results and Problems in Cell Differentiation, Vol. 4, ed. W. Beermann (Springer-Verlag, Berlin--Heidelberg--New York) (1972). Staub, M. : Chromosoma 26, 76--104 (1969). Stent, G.S.: Molecular genetics and introductory narrative (W.H. Freeman and Co., San Francisco) (1971). Zhimulev, I.F. : Chromosoma 46, 59--76 (1974). Zhimulev, I.F. and E.S. Belyaeva: Chromosoma 4 9 , 2 1 9 - - 2 3 1 (1975).